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PDF file, 19KB, TUNEL staining of BTC cell lines untreated (NT) and treated (SAR) with Saracatinib at the concentration of 5 �M for 24 hours. A statistical significant increase of apoptosis was seen in all the cell lines ( p-value < 0.05) except in HuH28.

PDF file, 21KB, Ki-67 staining of tumor tissue sections derived from vehicle (A) and Saracatinib treated group (B). A statistical significant decrease of proliferation was seen in treated mice (C) (p-value = 0.0003). NT: vehicle-treated mice.

PDF file, 28KB, Statistical analysis of reduction of cell proliferation by saracatinib in BTC cell lines. A statistical significant inhibition was revealed up to 1.25 �M in all the cell lines, except for TGBC1, in which no significance was found. In x-axis, doses of Saracatinib. In y-axis, absorbance.

PDF file, 9KB, Basal activated Src expression in BTC cell lines.

PDF file, 548KB, Differentially expressed genes obtained in in vitro and in vivo experiments; Saracatinib-deregulated genes both in vitro and in vivo models of BTC.

PDF file, 9KB, Inhibition of cellular foci formation: Saracatinib had the ability to reduce the formation of cellular foci in all the cell lines at Dm of 1 μM after 72 hours of treatment.

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PDF file, 61KB, Clinical pathological characteristics of BTC patients.

Biliary tract carcinoma (BTC) has a poor prognosis due to limited treatment options. There is, therefore, an urgent need to identify new targets and to design innovative therapeutic approaches. Among potential candidate molecules, we evaluated the nonreceptor tyrosine kinase Src, observing promising antitumor effects of its small-molecule inhibitor saracatinib in BTC preclinical models. The presence of an active Src protein was investigated by immunohistochemistry in 19 surgical samples from patients with BTC. Upon saracatinib treatment, the phosphorylation of Src and of its downstream transducers was evaluated in the BTC cell lines TFK-1, EGI-1, HuH28, and TGBC1-TKB. The effect of saracatinib on proliferation and migration was analyzed in these same cell lines, and its antitumor activity was essayed in EGI-1 mouse xenografts. Saracatinib-modulated transcriptome was profiled in EGI-1 cells and in tumor samples of the xenograft model. Src was activated in about 80% of the human BTC samples. In cultured BTC cell lines, low-dose saracatinib counteracted the activation of Src and of its downstream effectors, increased the fraction of cells in G0–G1 phase, and inhibited cell migration. At high concentrations (median dose from 2.26–6.99 μmol/L), saracatinib was also capable of inhibiting BTC cell proliferation. In vivo, saracatinib treatment resulted in delayed tumor growth, associated with an impaired vascular network. Here, we provide a demonstration that the targeted inhibition of Src kinase by saracatinib is of therapeutic benefit in preclinical models of BTC. We propose our results as a basis for the design of saracatinib-based clinical applications. Mol Cancer Ther; 11(7); 1528–38. ©2012 AACR.

PDF file, 11KB, Representative images of p-Src expression in BTC case series. A: Negative control. B-C-D: p-Src expression scored 1+, 2+ and 3+ respectively.

Background and aims: Cholangiocarcinoma (CCA) is a form of primary liver cancer with limited therapeutic options. Recently, cancer stem cells (CSCs) have been proposed as a driving force of tumour initiation and dissemination, thus representing a crucial therapeutic target. The protease inhibitor SerpinB3 (SB3) has been identified in several malignancies including hepatocellular carcinoma. SB3 has been involved in the early events of hepatocarcinogenesis and is highly expressed in hepatic progenitor cells and in a mouse model of liver progenitor cell activation. However, only limited information on the possible role of SB3 in CCA stem-like compartment is available. Methods: Enrichment of CCA stem-like subset was performed by sphere culture (SPH) in CCA cell lines (CCLP1, HUCCT1, MTCHC01 and SG231). Quantitative RT-PCR and Western blotting were used to detect SB3 in both SPH and parental monolayer (MON) cells. Acquired CSC-like features were analysed using an endogenous and a paracrine in vitro model, with transfection of SB3 gene or addition of recombinant SB3 to cell medium respectively. SB3 tumorigenic role was explored in an in vivo mouse model of CCA by subcutaneous injection of SB3-transfected MON (MONSB3+) cells in immune-deficient NOD-SCID/IL2Rgnull (NSG) mice. SB3 expression in human CCA sections was investigated by immunohistochemistry. Overall survival (OS) and time to recurrence (TTR) analyses were carried out from a transcriptome database of 104 CCA patients. Results: SB3, barely detected in parental MON cells, was overexpressed in the same CCA cells grown as 3D SPH. Notably, MONSB3+ showed significant overexpression of genes associated with stemness (CD24, CD44, CD133), pluripotency (c-MYC, NOTCH1, STAT3, YAP, NANOG, BMI1, KLF4, OCT4, SOX2), epithelial mesenchymal transition (β-catenin, SLUG) and extracellular matrix remodelling (MMP1, MMP7, MMP9, ADAM9, ADAM10, ADAM17, ITGB3). SB3-overexpressing cells showed superior spherogenic capacity and invasion ability compared to control. Importantly, MONSB3+ exhibited activation of MAP kinases (ERK1/2, p38, JNK) as well as phosphorylation of NFκB (p65) in addition to up-regulation of the proto-oncogene β-catenin. All these effects were reversed after transient silencing of SB3. According to the in vitro finding, MONSB3+ cells retained high tumorigenic potential in NSG mice. SB3 overexpression was observed in human CCA tissues and analysis of OS as well as TTR indicated a worse prognosis in SB3+ CCA patients. Conclusion: These findings indicate a SB3 role in mediating malignant phenotype of CCA and identify a new therapeutic target.

Chemotherapy resistance is a relevant clinical issue in tumor treatment, in particular in biliary tract carcinoma (BTC), for which there are no effective therapies, neither in the first nor in the second line. The development of chemoresistant cell lines as experimental models to investigate the mechanisms of resistance and identify alternative druggable pathways is mandatory. In BTC, in which genetics and biological behavior depend on the etiology, ethnicity, and anatomical site of origin, the creation of models that better recapitulate these characteristics is even more crucial. Here we have established and characterized an intrahepatic cholangiocarcinoma (iCCA) cell line derived from an Italian patient, called 82.3. Cells were isolated from a patient-derived xenograft (PDX) and, after establishment, immunophenotypic, biological, genetic, molecular characteristics, and tumorigenicity in vivo in NOD/SCID mice were investigated. 82.3 cells exhibited epithelial morphology and cell markers (EPCAM, CK7, and CK19), they also expressed different cancer stem markers (CD44, CD133, CD49b, CD24, Stro1, PAX6, FOXA2, OCT3/4), α–fetoprotein and under anchorage-independent and serum-free conditions were capable of originating cholangiospheres. The population doubling time was approximately 53 h. In vitro, they demonstrated a poor ability to migrate, in vivo, 82.3 cells retained their tumorigenicity, with a long latency period (16 weeks). Genetic identity using DNA fingerprinting analysis revealed 16 different loci, and the cell line was characterized by a complex hyperdiploid karyotype. Furthermore, 82.3 cells showed cross-resistance to gemcitabine, 5-fluorouracil, carboplatin, and oxaliplatin, in fact, their genetic profile showed that 60% of genes (n = 168), specific for drug resistance and related to the epithelial-mesenchymal transition, were deregulated in 82.3 cells compared to a control iCCA cell line sensitive to chemotherapeutics. RNA sequencing analysis revealed the enrichment for genes associated with epithelial to mesenchymal transition (EMT), vasculature development, and extracellular matrix (ECM) remodeling, underlining an aggressive phenotype. In conclusion, we have created a new iCCA cell line of Caucasian origin: this could be exploited as a preclinical model to study drug resistance mechanisms and to identify alternative therapies to improve the prognosis of this tumor type.

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Cholangiocarcinoma (CCA) is a rare, aggressive disease with poor overall survival. In advanced cases, surgery is often not possible or fails; in addition, there is a lack of effective and specific therapies. Multidisciplinary approaches and advanced technologies have improved the knowledge of CCA molecular pathogenesis, highlighting its extreme heterogeneity and high frequency of genetic and molecular aberrations. Effective preclinical models, therefore, should be based on a comparable level of complexity. In the past years, there has been a consistent increase in the number of available CCA models. The exploitation of even more complex CCA models is rising. Examples are the use of CRISPR/Cas9 or stabilized organoids for in vitro studies, as well as patient-derived xenografts or transgenic mouse models for in vivo applications. Here, we examine the available preclinical CCA models exploited to investigate: (i) carcinogenesis processes from initiation to progression; and (ii) tools for personalized therapy and innovative therapeutic approaches, including chemotherapy and immune/targeted therapies. For each model, we describe the potential applications, highlighting both its advantages and limits.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive and lethal malignancy with limited therapeutic options. Trabectedin has a high antitumor activity in preclinical models of biliary tract carcinoma (BTC), being a promising alternative treatment. Here, we studied the effect of trabectedin at transcriptomic level on an ICC patient derived xenograft (PDX) and on the derived cell line, MT-CHC01. Further, putative targets of trabectedin were explored in the in vitro model. In vitro, trabectedin inhibited genes involved in protein modification, neurogenesis, migration, and motility; it induced the expression of genes involved in keratinization, tissues development, and apoptotic processes. In the PDX model, trabectedin affected ECM-receptor interaction, focal adhesion, complement and coagulation cascades, Hedgehog, MAPK, EGFR signaling via PIP3 pathway, and apoptosis. Among down-regulated genes, we selected SYK and LGALS1; their silencing caused a significantly reduction of migration, but did not affect proliferation in in vitro models. In MT-CHC01 cells, 24 microRNAs were deregulated upon drug treatment, while only 5 microRNAs were perturbed by trabectedin in PDX. The target prediction analysis showed that SYK and LGALS1 are putative targets of up-regulated microRNAs. In conclusion, we described that trabectedin affected genes and microRNAs involved in tumor progression and metastatic processes, reflecting data previously obtained at macroscopically level; in particular, we identified SYK and LGALS1 as new putative targets of trabectedin.

Background Fluoropyrimidines (FP) are the backbone chemotherapy (CT) for colorectal cancer (CRC). Although the most common toxicities have been extensively studied, FP-induced cardiotoxicity (FIC), an infrequent but potentially life-threatening toxicity, still lacks of a comprehensive characterization. The correlation between FIC and known cardiovascular (CV) risk factors remains controversial and based on retrospective observations. Methods CRC patients (pts) treated for the first time with FP at Candiolo Cancer Institute have been enrolled since January 2016. All pts were screened for potential CV risk factors; if needed, the treatment of preexisting CV comorbidities was optimized before starting CT. During the first 3 CT cycles, a monitoring with CV symptoms collection, seriated electrocardiograms and brain natriuretic peptide (BNP) measurements was performed. Primary objective was to assess the incidence of FIC. Secondary objectives included the analysis of the relationship of FIC with known CV risk factors and BNP levels. Results An interim analysis was conducted on 101 pts (65% male, median age 71.6 years). We found high prevalence of CV risk factors (BMI ≥25 54.2%, smoker 50%, heavy drinker 23%, sedentary lifestyle 63.3%) and comorbidities (diabetes mellitus 19.2%, dyslipidemia 34.7%, arterial hypertension 54%, stroke 3%, coronary artery disease 6%, arrhythmias 8%, heart failure 4%). 19 pts (18.8%) experienced FIC: 1 acute coronary syndrome (ACS), 1 coronary vasospasm, 1 paroxysmal supraventricular tachycardia (PSVT), 1 complete left bundle branch block (LBBB), 2 syncope, 4 typical chest pain, 6 sudden dyspnea, 3 sudden palpitations. After treatment of the CV events, only 3 pts had to discontinue FP (ACS, LBBB and PSTV). Among symptomatic pts, only 47% had CV comorbidities and/or CV risk factors. BNP levels increased on average by 73% [CI 99%: +39% - +106%] after the first cycle. Conclusions A high incidence of CV events, with no apparent correlation with CV comorbidities or risk factors, was observed. Prompt identification and treatment of CV events allowed most pts to complete the treatment with FP. Clinical trial identification NCT02665312. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure M. Aglietta: Advisory / Consultancy: Bristol-Myers Squibb; Advisory / Consultancy: Merck; Advisory / Consultancy: Roche. All other authors have declared no conflicts of interest.

Intrahepatic cholangiocarcinoma (ICC) is one of the most lethal liver cancers. Late diagnosis and chemotherapy resistance contribute to the scarce outfit and poor survival. Resistance mechanisms are still poorly understood. Here, we established a Gemcitabine (GEM) resistant model, the MT-CHC01R1.5 cell line, obtained by a GEM gradual exposure (up to 1.5 &micro, M) of the sensitive counterpart, MT-CHC01. GEM resistance was irreversible, even at high doses. The in vitro and in vivo growth was slower than MT-CHC01, and no differences were highlighted in terms of migration and invasion. Drug prediction analysis suggested that Paclitaxel and Doxycycline might overcome GEM resistance. Indeed, in vitro MT-CHC01R1.5 growth was reduced by Paclitaxel and Doxycycline. Importantly, Doxycycline pretreatment at very low doses restored GEM sensitivity. To assess molecular mechanisms underlying the acquisition of GEM resistance, a detailed analysis of the transcriptome in MT-CHC01R1.5 cells versus the corresponding parental counterpart was performed. Transcriptomic analysis showed that most up-regulated genes were involved in cell cycle regulation and in the DNA related process, while most down-regulated genes were involved in the response to stimuli, xenobiotic metabolism, and angiogenesis. Furthermore, additional panels of drug resistance and epithelial to mesenchymal transition genes (n = 168) were tested by qRT-PCR and the expression of 20 genes was affected. Next, based on a comparison between qRT-PCR and microarray data, a list of up-regulated genes in MT-CHC01R1.5 was selected and further confirmed in a primary cell culture obtained from an ICC patient resistant to GEM. In conclusion, we characterized a new GEM resistance ICC model that could be exploited either to study alternative mechanisms of resistance or to explore new therapies.

Hotspot codon 132 mutations (R132xIDH1m) are frequent in intrahepatic cholangiocarcinoma (ICC), are druggable by anti-IDH1m agents, and could represent a marker of disease progression. Developing an assay to identify R132xIDH1m would provide a useful tool to select patients benefitting from targeted treatments. We tested a quantitative real-time allele-specific polymerase chain reaction (qPCR)-based method to detect the main R132xIDH1m in an Italian ICC series (n = 61) of formalin-fixed paraffin-embedded (FFPE) samples, and on circulating-free DNA samples. The outcomes were compared with nested PCR/Sanger sequencing. Reconstitution experiments of plasmids harboring the different R132xIDH1m mixed with wild-type (WT) DNA demonstrated that qPCR is able to detect at least 2% of all mutated allele. High efficiency was also observed on patient-derived mutated DNA mixed with WT DNA (up to 10% and 0.3 ng of mutated template), qPCR detected 16.4% of mutated samples (one R132G, three R132C and six R132L) while nested PCR/Sanger sequencing only 8.2% (four R132L and one R132G). In a single patient with an R132C-mutated tumor, qPCR was also performed on plasma samples collected at four time-points, observing an increase correlating with disease progression. In conclusion, we developed a qPCR assay which could represent a fast, inexpensive and sensitive tool both for detection of R132xIDH1m in ICC samples and monitoring disease progression from liquid biopsy.

Background Effective target therapies for intrahepatic cholangiocarcinoma (ICC) have not been identified so far. One of the reasons may be the genetic evolution from primary (PR) to recurrent (REC) tumors. We aim to identify peculiar characteristics and to select potential targets specific for recurrent tumors. Eighteen ICC paired PR and REC tumors were collected from 5 Italian Centers. Eleven pairs were analyzed for gene expression profiling and 16 for mutational status of IDH1. For one pair, deep mutational analysis by Next Generation Sequencing was also carried out. An independent cohort of patients was used for validation. Results Two class-paired comparison yielded 315 differentially expressed genes between REC and PR tumors. Up-regulated genes in RECs are involved in RNA/DNA processing, cell cycle, epithelial to mesenchymal transition (EMT), resistance to apoptosis, and cytoskeleton remodeling. Down-regulated genes participate to epithelial cell differentiation, proteolysis, apoptotic, immune response, and inflammatory processes. A 24 gene signature is able to discriminate RECs from PRs in an independent cohort; FANCG is statistically associated with survival in the chol-TCGA dataset. IDH1 was mutated in the RECs of five patients; 4 of them displayed the mutation only in RECs. Deep sequencing performed in one patient confirmed the IDH1 mutation in REC. Conclusions RECs are enriched for genes involved in EMT, resistance to apoptosis, and cytoskeleton remodeling. Key players of these pathways might be considered druggable targets in RECs. IDH1 is mutated in 30% of RECs, becoming both a marker of progression and a target for therapy.

The association of anti-EGFR to gemcitabine and oxaliplatin (GEMOX) chemotherapy did not improve survival in biliary tract carcinoma (BTC) patients. Multiple mechanisms might be involved in the resistance to anti-EGFR. Here, we explored the mutation profile of EGFR extracellular domain (ECD), of tyrosine kinase domain (TKD), and its amplification status. EGFR mutational status of exons 12, 18–21 was analyzed in 57 tumors by Sanger sequencing. EGFR amplification was evaluated in 37 tumors by Fluorescent In Situ Hybridization (FISH). Kaplan-Meier curves were calculated using the log-rank test. Six patients had mutations in exon 12 of EGFR ECD and 7 in EGFR TKD. Neither EGFR ECD nor TKD mutations affected progression free survival (PFS) or overall survival (OS) in the entire population. In the panitumumab plus GEMOX (P-GEMOX) arm, ECD mutated patients had a worse OS, while EGFR TKD mutated patients had a trend towards shorter PFS and OS. Overall, the presence of mutations in EGFR or in its transducers did not affect PFS or OS, while the extrahepatic cholangiocarcinoma (ECC) mutated patients had a worse prognosis compared to WT. Nineteen out of 37 tumors were EGFR amplified, but the amplification did not correlate with survival. ECC EGFR amplified patients had improved OS, whereas the amplification significantly correlated with poor PFS (p = 0.03) in gallbladder carcinoma patients. The high molecular heterogeneity is a predominant feature of BTC: the alterations found in this work seem to have a prognostic impact rather than a predictive role towards anti-EGFR therapy.

ROS1 rearrangements have been detected in a variety of tumors and are considered as suitable targets of anticancer therapies. We developed a new, quick, specific, and sensitive PCR test to screen for the FIG-ROS1 fusion and applied it to a series of Italian patients with bile duct carcinoma (BTC). Formalin-fixed, paraffin-embedded tissues, derived from 65 Italian BTC patients, and six cell lines were analyzed by nested PCR to investigate the prevalence of a previously reported FIG-ROS1 fusion. The specificity and sensitivity of nested PCR were investigated in FIG-ROS1 positive U118MG cells in reconstitution experiments with peripheral blood mononuclear cells. We found that six out of 65 (9%) BTC patients were positive for the FIG-ROS1 fusion, comprising two out of 14 (14%) gallbladder carcinoma (GBC) patients and four out of 25 (16%) extrahepatic cholangiocarcinoma (ECC) patients. None of the 26 intrahepatic cholangiocarcinoma cases harbored the FIG-ROS1 fusion. All the cell lines were negative for this variant. In conclusion, 14-16% of GBC and ECC were positive for FIG-ROS1. This may have clinical implications, since these patients will potentially benefit from the treatment with specific ROS1 inhibitors.

Biliary tract carcinomas (BTC) are a group of tumours arising from the epithelial cells of intra- and extra-hepatic biliaryducts and the gallbladder, characterised by a poor prognosis. Surgery is the only curative procedure, but the risk of recurrence is high and furthermore, the majority of patients present with unresectable disease at the time of diagnosis. Systemic therapy is the mainstay of treatment for patients who present recurrent or metastatic disease. Progress has been made in the last decade to identify the most effective chemotherapy regimens, with the recent recommendation of the combination of gemcitabine–cisplatin as the standard schedule. Comprehension of the molecular basis of cholangiocarcinogenesis and tumour progression has recently led to the experimentation of targeted therapies in patients with BTC, demonstrating promising results. In this review we will discuss the clinical experience with systemic treatment for BTC, focusing on future directions with targeted therapies. © 2012 Elsevier Ireland Ltd. All rights reserved.

Biliary tract carcinomas (BTC) are malignant tumors with limited therapeutic options. Clinical experiences with anti-EGFR therapies have produced unsatisfactory results. The strategies of combined inhibition of EGFR and MEK1/2 could be a promising therapeutic option in BTC treatment. Preclinical activity of Panitumumab and Trametinib was tested in in vitro (EGI-1, MT-CHC01 and WITT cells) and in in vivo (xenograft) BTC models with different K-RAS mutational status. Trametinib reduced MAPK phosphorylation in wild type (WT) WITT cells and in both K-RAS mutated cells; in EGI-1 was also able to switch off EGFR activation. Panitumumab reduced the activation of its target only in EGI-1 cells, and of MAPK only in WITT cells. While Trametinib inhibited cell growth in K-RAS mutated cell lines, Panitumumab had no effect on proliferation independently by K-RAS status. The addition of Panitumumab to Trametinib did not significantly potentiate its anti-proliferative effect also in mutated cells. In vivo, Trametinib was able to significantly slow the tumor growth in K-RAS mutated xenograft models, but did not have effect on K-RAS WT cells; the addition of Panitumumab potentiated the Trametinib efficacy in MT-CHC01 and overcame the resistance to the anti-EGFR in WITT cells, in which the monotherapy was ineffective. Only in K-RAS mutated xenografts Trametinib alone or in combination with Panitumumab significantly decreased Ki67 positive cell fraction and CD31 angiogenesis markers. In conclusion, this preclinical study provides a rational to plan clinical trials assessing the efficacy of Trametinib in K-RAS mutated BTC patients and the combination with anti-EGFR in WT BTC patients.

Biliary tract carcinoma is a rare malignancy with multiple causes, which underlie the different genetic and molecular profiles. Cancer cell lines are affordable models, reflecting the characteristics of the tumor of origin. They represent useful tools to identify molecular targets for treatment. Here, we established and characterized from biological, molecular, and genetic point of view, an Italian intrahepatic cholangiocarcinoma cell line (ICC), the MT-CHC01. MT-CHC01 cells were isolated from a tumor-derived xenograft. Immunophenotypical characterization was evaluated both at early and after stabilization passages. In vitro biological, genetic, and molecular features were also investigated. In vivo tumorigenicity was assessed in NOD/SCID mice. MT-CHC01cells retain epithelial cell markers, EPCAM, CK7, and CK19, and some stemness and pluripotency markers, i.e., SOX2, Nanog, CD49f/integrin-α6, CD24, PDX1, FOXA2, and CD133. They grow as a monolayer, with a population double time of about 40 h; they show a low migration and invasion potential. In low attachment conditions, they are able to form spheres and to growth in anchorage-independent manner. After subcutaneous injection, they retain in vivo tumorigenicity; the expression of biliary markers as CA19-9 and CEA were maintained from primary tumor. The karyotype is highly complex, with a hypotriploid to hypertriploid modal number (3n+/−) (52 to 77 chromosomes); low level of HER2 gene amplification, TP53 deletion, gain of AURKA were identified; K-RAS G12D mutation were maintained from primary tumor to MT-CHC01 cells. We established the first ICC cell line derived from an Italian patient. It will help to study either the biology of this tumor or to test drugs both in vitro and in vivo. Electronic supplementary material The online version of this article (doi:10.1007/s13277-015-4215-3) contains supplementary material, which is available to authorized users.

Background Standard chemotherapy in unresectable biliary tract carcinoma (BTC) patients is based on gemcitabine combined with platinum derivatives. However, primary or acquired resistance is inevitable and no second-line chemotherapy is demonstrated to be effective. Thus, there is an urgent need to identify new alternative (chemo)therapy approaches. Methods We evaluated the mechanism of action of ET-743 in preclinical models of BTC. Six BTC cell lines (TFK-1, EGI-1, TGBC1, WITT, KMCH, HuH28), two primary cell cultures derived from BTC patients, the EGI-1 and a new established BTC patient-derived xenografts, were used as preclinical models to investigate the anti-tumor activity of ET-743 in vitro and in vivo. Gene expression profiling was also analyzed upon ET-743 treatment in in vivo models. Results We found that ET-743 inhibited cell growth of BTC cell lines and primary cultures (IC50 ranging from 0.37 to 3.08 nM) preferentially inducing apoptosis and activation of the complex DNA damage-repair proteins (p-ATM, p-p53 and p-Histone H2A.x) in vitro. In EGI-1 and patient-derived xenografts, ET-743 induced tumor growth delay and reduction of vasculogenesis. In vivo ET-743 induced a deregulation of genes involved in cell adhesion, stress-related response, and in pathways involved in cholangiocarcinogenesis, such as the IL-6, Sonic Hedgehog and Wnt signaling pathways. Conclusions These results suggest that ET-743 could represent an alternative chemotherapy for BTC treatment and encourage the development of clinical trials in BTC patients resistant to standard chemotherapy. Electronic supplementary material The online version of this article (doi:10.1186/1471-2407-14-918) contains supplementary material, which is available to authorized users.

The HER2/c-ErbB-2 proto-oncogene is overexpressed in 25–30% of human breast cancers. We previously reported the c-ErbB-2 transcript in mononuclear cells (MNC) from bone marrow (BM), peripheral blood (PB), and mobilized PB (MPB). Here, we describe extensively the expression pattern of c-ErbB-2 mRNA and protein in normal adult hematopoietic tissue and cord blood (CB)-derived cells. Quantitative reverse transcriptase-polymerase chain reaction shows that the c-ErbB-2 transcript is expressed in hematopoietic cells at low levels if compared with normal epithelial and breast cancer cells. The c-ErbB-2 protein was detected predominantly in MNC from PB and CB by Western blot analysis. Flow cytometry revealed that CD15+, CD14+, and glycophorin A+ subpopulations express c-ErbB-2 protein, whereas lymphocytes are c-ErbB-2-negative. The c-ErbB-2 expression is higher in CB MNC. More than 90% of BM- and MPB-derived CD34+ progenitors are c-ErbB-2-negative; by contrast, 5–40% of CB-derived CD34+ progenitors express c-ErbB-2. We found that c-ErbB-2 protein is up-regulated during cell-cycle recruitment of progenitor cells. Similarly, it increases in mature, hematopoietic proliferating cells. This study reports the first evidence that the c-ErbB-2 receptor is correlated to the proliferating state of hematopoietic cells. Studies in progress aim to clarify the role of c-ErbB-2 in regulation of this process in hematopoietic tissues.

ROS1 rearrangements have been detected in a variety of tumors and are considered as suitable targets of anticancer therapies. We developed a new, quick, specific, and sensitive PCR test to screen for the FIG-ROS1 fusion and applied it to a series of Italian patients with bile duct carcinoma (BTC). Formalin-fixed, paraffin-embedded tissues, derived from 65 Italian BTC patients, and six cell lines were analyzed by nested PCR to investigate the prevalence of a previously reported FIG-ROS1 fusion. The specificity and sensitivity of nested PCR were investigated in FIG-ROS1 positive U118MG cells in reconstitution experiments with peripheral blood mononuclear cells. We found that six out of 65 (9%) BTC patients were positive for the FIG-ROS1 fusion, comprising two out of 14 (14%) gallbladder carcinoma (GBC) patients and four out of 25 (16%) extrahepatic cholangiocarcinoma (ECC) patients. None of the 26 intrahepatic cholangiocarcinoma cases harbored the FIG-ROS1 fusion. All the cell lines were negative for this variant. In conclusion, 14-16% of GBC and ECC were positive for FIG-ROS1. This may have clinical implications, since these patients will potentially benefit from the treatment with specific ROS1 inhibitors.

Identification of culture conditions that support expansion or even long-term maintenance of in vivo repopulating human hematopoietic stem cells is still a major challenge. Using a combination of FLT3 ligand (FL), Stem Cell Factor (SCF), Thrombopoietin (TPO) and Interleukin 6 (IL6), we cultured cord blood (CB) CD34+ cells for up to 12 weeks and transplanted their progeny into sublethally irradiated NOD/SCID mice. Bone marrow engraftment was considered successful when recipients contained measurable numbers of human CD45+, CD71+ and Glycophorin A+(GpA) cells 8 weeks after transplantation. Twelve-week expanded cells with FL+SCF+TPO+IL6 successfully engrafted all of the recipients and human CD45(+)+CD71(+)+GpA(+) cells represented 4.3 to 22.4% of bone marrow. Substitution of IL6 with IL3 led to an even better expansion of cells and a similar clonogenic progenitor output in the first 8 weeks of culture; however, LTC-IC output increased up to week 6 and then decreased and disappeared. By contrast, with FL+SCF+TPO+IL6, LTC-IC kept increasing up to week 12. Four-week cultured cells with FL+SCF+TPO+IL3 less efficiently engrafted NOD/SCID mice, both as measured by frequency of positive recipients (4 out of 10) and percentage of engrafted human cells (< or =2%). Six-week expanded cells failed to engraft. This study provides evidence that many, but not all, of the so-called "early acting" cytokines, can sustain long-term maintenance and even expansion of human primitive in vivo repopulating stem cells. In particular, in the culture conditions used in this study, the presence of IL3 greatly reduces the repopulating potential of expanded CD34+ CB cells.

Understanding the repopulating characteristics of human hematopoietic stem/progenitor cells is crucial for predicting their performance after transplant into patients receiving high-dose radiochemotherapy. We have previously reported that CD34+cord blood (CB) cells can be expanded in vitro for several months in serum containing culture conditions. The use of combinations of recombinant early acting growth factors and the absence of stroma was essential in determining this phenomenon. However, the effect of these manipulations on in vivo repopulating hematopoietic cells is not known. Recently, a new approach has been developed to establish an in vivo model for human primitive hematopoietic precursors by transplanting human hematopoietic cells into sublethally irradiated nonobese diabetic severe combined immunodeficient (NOD/SCID) mice. We have examined here the expansion of cells, CD34+ and CD34+38− subpopulations, colony-forming cells (CFC), long-term culture initiating cells (LTC-IC) and the maintenance or the expansion of SCID-repopulating cells (SRC) during stroma-free suspension cultures of human CD34+ CB cells for up to 12 weeks. Groups of sublethally irradiated NOD/SCID mice were injected with either 35,000, 20,000, and 10,000 unmanipulated CD34+ CB cells, which were cryopreserved at the start of cultures, or the cryopreserved cells expanded from 35,000, 20,000, or 10,000 CD34+ cells for 4, 8, and 12 weeks in the presence of a combination of early acting recombinant growth factors (flt 3/flk2 ligand [FL] + megakaryocyte growth and development factor [MGDF] ± stem cell factor [SCF] ± interleukin-6 [IL-6]). Mice that had been injected with ≥20,000 fresh or cryopreserved uncultured CD34+ cells did not show any sign or showed little engraftment in a limited number of animals. Conversely, cells that had been generated by the same number of initial CD34+ CB cells in 4 to 10 weeks of expansion cultures engrafted the vast majority of NOD/SCID mice. The level of engraftment, well above that usually observed when the same numbers of uncultured cells were injected in the same recipients (even in the presence of irradiated CD34− cells) suggested that primitive hematopoietic cells were maintained for up to 10 weeks of cultures. In addition, dilution experiments suggest that SRC are expanded more than 70-fold after 9 to 10 weeks of expansion. These results support and extend our previous findings that CD34+ CB stem cells (identified as LTC-IC) could indeed be grown and expanded in vitro for an extremely long period of time. Such information may be essential to design efficient stem cell expansion procedures for clinical use.

Biliary tract carcinoma (BTC) has a poor prognosis due to limited treatment options. There is, therefore, an urgent need to identify new targets and to design innovative therapeutic approaches. Among potential candidate molecules, we evaluated the nonreceptor tyrosine kinase Src, observing promising antitumor effects of its small-molecule inhibitor saracatinib in BTC preclinical models. The presence of an active Src protein was investigated by immunohistochemistry in 19 surgical samples from patients with BTC. Upon saracatinib treatment, the phosphorylation of Src and of its downstream transducers was evaluated in the BTC cell lines TFK-1, EGI-1, HuH28, and TGBC1-TKB. The effect of saracatinib on proliferation and migration was analyzed in these same cell lines, and its antitumor activity was essayed in EGI-1 mouse xenografts. Saracatinib-modulated transcriptome was profiled in EGI-1 cells and in tumor samples of the xenograft model. Src was activated in about 80% of the human BTC samples. In cultured BTC cell lines, low-dose saracatinib counteracted the activation of Src and of its downstream effectors, increased the fraction of cells in G0–G1 phase, and inhibited cell migration. At high concentrations (median dose from 2.26–6.99 μmol/L), saracatinib was also capable of inhibiting BTC cell proliferation. In vivo, saracatinib treatment resulted in delayed tumor growth, associated with an impaired vascular network. Here, we provide a demonstration that the targeted inhibition of Src kinase by saracatinib is of therapeutic benefit in preclinical models of BTC. We propose our results as a basis for the design of saracatinib-based clinical applications. Mol Cancer Ther; 11(7); 1528–38. ©2012 AACR.

Background Activating mutations of the epidermal growth factor receptor (EGFR) confer sensitivity to the tyrosine kinase inhibitors (TKi), gefitinib and erlotinib. We analysed EGFR expression, EGFR mutation status and gene expression profiles of prostate cancer (PC) to supply a rationale for EGFR targeted therapies in this disease. Methods Mutational analysis of EGFR TK domain (exons from 18 to 21) and immunohistochemistry for EGFR were performed on tumour tissues derived from radical prostatectomy from 100 PC patients. Gene expression profiling using oligo-microarrays was also carried out in 51 of the PC samples. Results EGFR protein overexpression (EGFRhigh) was found in 36% of the tumour samples, and mutations were found in 13% of samples. Patients with EGFRhigh tumours experienced a significantly increased risk of biochemical relapse (hazard ratio-HR 2.52, p=0.02) compared with patients with tumours expressing low levels of EGFR (EGFRlow). Microarray analysis did not reveal any differences in gene expression between EGFRhigh and EGFRlow tumours. Conversely, in EGFRhigh tumours, we were able to identify a 79 gene signature distinguishing mutated from non-mutated tumours. Additionally, 29 genes were found to be differentially expressed between mutated/EGFRhigh (n=3) and mutated/EGFRlow tumours (n=5). Four of the down-regulated genes, U19/EAF2, ABCC4, KLK3 and ANXA3 and one of the up-regulated genes, FOXC1, are involved in PC progression. Conclusions Based on our findings, we hypothesize that accurate definition of the EGFR status could improve prognostic stratification and we suggest a possible role for EGFR-directed therapies in PC patients. Having been generated in a relatively small sample of patients, our results warrant confirmation in larger series.

Trastuzumab has changed the prognosis of HER2 positive breast cancers. Despite this progress, resistance to trastuzumab occurs in most patients. Newer anti-HER2 therapies, like the dual tyrosine-kinase inhibitor (TKI) lapatinib, show significant antitumor activity, indicating that HER2 can be still exploited as a target after trastuzumab failure. However, since a high proportion of patients fail to respond to these alternative strategies, it is possible that cell escape from HER2 targeting may rely on HER2 independent pathways. The knowledge of these pathways deserve to be exploited to develop new therapies. We characterized two human HER2 overexpressing breast cancer cell lines resistant to trastuzumab and lapatinib (T100 and JIMT-1) from a molecular and biological point of view. Indeed, we assessed both in vitro and in vivo the activity of the multitarget inhibitor sorafenib. In both cell lines, the previously proposed mechanisms did not explain resistance to HER2 inhibitors. Notably, silencing HER2 by shRNA did not affect the growth of our cells, suggesting loss of reliance upon HER2. Moreover, we identified alterations in two antiapoptotic proteins Mcl-1 and Survivin which are known to be targets of the multikinase inhibitor sorafenib. Moreover, sorafenib, strongly inhibited the in vitro growth of T100 and JIMT-1 cells, through the downregulation of both Mcl-1 and Survivin. Similar results were obtained in JIMT-1 xenografts subcutaneously injected in NOD SCID mice. We provide preclinical evidence that tumor cells resistant to trastuzumab and lapatinib may rely on HER2 independent pathways that can be efficiently inhibited by sorafenib.

Background Advanced biliary tract carcinomas (BTCs) have poor prognosis and limited therapeutic options. Therefore, it is crucial to combine standard therapies with molecular targeting. In this study EGFR, HER2, and their molecular transducers were analysed in terms of mutations, amplifications and over-expression in a BTC case series. Furthermore, we tested the efficacy of drugs targeting these molecules, as single agents or in combination with gemcitabine, the standard therapeutic agent against BTC. Methods Immunohistochemistry, FISH and mutational analysis were performed on 49 BTC samples of intrahepatic (ICCs), extrahepatic (ECCs), and gallbladder (GBCs) origin. The effect on cell proliferation of different EGFR/HER2 pathway inhibitors as single agents or in combination with gemcitabine was investigated on BTC cell lines. Western blot analyses were performed to investigate molecular mechanisms of targeted drugs. Results EGFR is expressed in 100% of ICCs, 52.6% of ECCs, and in 38.5% of GBCs. P-MAPK and p-Akt are highly expressed in ICCs (>58% of samples), and to a lower extent in ECCs and GBCs ( Conclusion These data demonstrate that EGFR and HER2 pathways are suitable therapeutic targets for BTCs. The combination of gemcitabine with drugs targeting these pathways gives encouraging results and further clinical studies could be warranted.

The recombinant monoclonal antibody trastuzumab has antiproliferative effect on breast cancer (BC) cells with ErbB2 overexpression. We postulated that a mechanism able to modify ErbB2 expression enhances the antitumor effect of trastuzumab. We analyzed whether granulocyte-colony stimulating factor (G-CSF), widely used in adjuvant cancer therapy to alleviate chemotherapy-induced myelotoxicity, could influence ErbB2 expression in BC cells and patients. The expression of ErbB2 (Herceptest) was analyzed in four BC cell lines (BT474, SKBR3, ZR75.1, and T47D) treated with G-CSF and in five samples biopsies from BC patients subjected to G-CSF rescue after chemotherapy. The effects of G-CSF and trastuzumab alone or their combination on cell growth and apoptosis were investigated. G-CSF receptor was detected on all cell lines and BC patients. G-CSF induced upregulation of ErbB2 in SKBR3, ZR75, and T47D cells. This modulation was not associated with an increase in tumor cell growth in vitro. Trastuzumab alone inhibited colony formation in soft agar but did not induce apoptosis on BC cells with no or low ErbB2 genomic amplification. The combination of trastuzumab and G-CSF enhanced the inhibition of tumor colony formation and induced apoptosis on these cells. This effect was further increased by G-CSF pretreatment. Five of nine BC patients showed an increase of Herceptest score after G-CSF administration. G-CSF treatment increases ErbB2 expression in vitro and in vivo enhancing the activity of trastuzumab on BC cell lines inducing apoptosis of BC cells with low or no ErbB2 genomic amplification.

Intrahepatic cholangiocarcinoma (ICC) is the second most common liver cancer. Conventional chemotherapy and radiotherapy have only a marginal impact on the natural history with a median survival of 12 months. Encouraging results obtained on ICC cell lines prompted us to evaluate the antitumor activity and mechanism of action of ET-743 on an ICC patient-derived xenograft (PDX) model, currently representing an innovative tool to personalize cancer therapy. The antitumor activity of ET-743 was tested on a cohort of mice carrying an ICC PDX. At the achievement of about 150 mm3 of volume, mice were randomized in two arms (n=7) of therapy in three independent experiments: 150 mg/Kg/weekly i.v. of ET-743 or drug vehicle for three administrations. Tumors were monitored weekly. Mean volumes of treated and untreated xenografts were compared by two-way Anova, considering a p-value ET-743 slowed tumor growth of PDX-treated compared to PDX-untreated mice in a statistically significant manner (p=0.04). Interestingly, we found a decrease in the number of proliferating cells (p=0.05) and a significant decrease in the number of new vessel formation (p=0.007). Upon ET-743 treatment, 1346 differentially expressed probes were found: 628 down-regulated and 718 up-regulated in ET-743 treated vs control PDX mice. Gene Ontology analysis revealed that the most significant deregulated processes were biological and cell adhesion, tissue development and inflammatory/immune response. Among the 20 most down-regulated genes, we found TFF3, MUC5B, MUC6A, WISP1, previously associated with cholangiocarcinoma. miRNA profiling revealed a deregulation of 6 miRNAs, 3 up-regulated and 3 down-regulated. Using the Mirpath program, we found that the down-regulated miRNAs affect important cancer related pathways (p53, Akt-PI3K, MAPK pathways) in a statistically significant way. In conclusion, ET-743 is able to control tumor growth, inhibiting proliferation and neoangiogenesis; moreover ET-743 perturbs gene and miRNA profiling affecting important cancer related pathways. Taken together these data are a strong rational for the design of a clinical trial to evaluate the activity of ET-743 in ICC patients. Citation Format: Caterina Peraldo Neia, Giuliana Cavalloni, Giovanna Chiorino, Paola Ostano, Massimo Aglietta, Francesco Leone. Antitumor activity, gene and miRNA modulation upon ET-743 treatment in an intrahepatic cholangiocarcinoma patient-derived xenograft model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5551. doi:10.1158/1538-7445.AM2014-5551

Tumor cell contamination might induce relapse after autologous transplantation in breast cancer patients. We used an ex vivo purging strategy to decrease the number of contaminating breast tumor cells in leukaphereses without altering the engraftment potential of the hemopoietic progenitor cells. This method is based on immunoselection of CD34+ cells derived from mobilized peripheral blood of patients with metastatic breast cancer and expansion in the presence of flt3 ligand, stem cell factor, interleukin 6, and thrombopoietin. Tumor contamination before and after culture was monitored by mammaglobin messenger RNA amplification by quantitative polymerase chain reaction. We analyzed both adherent and suspended cells obtained after 2 weeks of culture. Hemopoietic progenitors were increased among suspended cells. In this fraction, tumor cell contamination was decreased, whereas it increased within the adherent cell fraction. Experimental models using CD34+ cells from healthy donors spiked with breast cancer cells were also constructed to investigate whether treatment with anti–ErbB-receptor drugs could further reduce the tumor load without affecting the clonogenic potential of hemopoietic cells. For this purpose, we successfully assayed trastuzumab, a monoclonal antibody against ErbB-2, and gefitinib, an epidermal growth factor receptor tyrosine kinase receptor inhibitor. These results suggest that positively selected CD34+ cells from cancer patients contain tumor cells and that ex vivo expansion can reduce the tumor load of the suspended fraction. Target-based agents against ErbB-2, epidermal growth factor receptor, or both—such as trastuzumab or gefitinib—might increase the efficiency of purging.

Objective Ex vivo expansion of human hemopoietic stem cells (HSC) is an important issue in transplantation and gene therapy. Encouraging results have been obtained with cord blood, where extensive amplification of primitive progenitors was observed. So far, this goal has been elusive with adult cells, in which amplification of committed and mature cells, but not of long-term repopulating cells, has been described. Methods Adult normal bone marrow (BM) and mobilized peripheral blood (MPB) CD34 + cells were cultured in a stroma-free liquid culture in the presence of Flt-3 ligand (FL), thrombopoietin (TPO), stem cell factor (SCF), interleukin-6 (IL-6), or interleukin-3 (IL-3). Suitable aliquots of cells were used to monitor cell production, clonogenic activity, LTC-IC output, and in vivo repopulating capacity. Results Here we report that BM and MPB HSC can be cultured in the presence of FL, TPO, SCF, and IL-6 for up to 10 weeks, during which time they proliferate and produce large numbers of committed progenitors (up to 3000-fold). Primitive NOD/SCID mouse repopulating stem cells (SRC) are expanded sixfold after 3 weeks (by limiting dilution studies) and retain the ability to repopulate secondary NOD/SCID mice after serial transplants. Substitution of IL-6 with IL-3 leads to a similarly high production of committed and differentiated cells but only to a transient (1 week) expansion of SRC s , which do not possess secondary repopulation capacity. Conclusion We report evidence to show that under appropriate culture conditions, adult human SRC can also be induced to expand with limited differentiation.

Identification of sensitive techniques for breast cancer cell detection might be relevant for high-dose chemotherapy programs with autologous stem cell transplantation. We investigated the feasibility of Maspin, Mammaglobin and c-ErbB-2 amplification by real-time quantitative polymerase chain reaction (RQ-PCR) for the detection of breast cancer cells in leukaphereses. Expression of the three markers was determined in primary breast cancers and cell lines. Peripheral blood (PB), bone marrow (BM), and leukapheresis samples from patients with malignancies other than breast cancer were used as controls. Sensitivity was evaluated by dilution of primary tumors and cell lines with mononuclear blood cells. We found expression of the three markers in all primary tumors and most cell lines. No blood specimen from control patients had the Maspin transcript, while only one was positive for Mammaglobin. Weak c-ErbB-2 expression was detectable in most PB, all BM and all leukapheresis samples from controls. We observed a low sensitivity of Maspin RQ-PCR and a sensitivity of Mammaglobin RQ-PCR up to one tumor cell in 106 mononuclear cells. One out of 18 leukaphereses from breast cancer patients screened for the presence of Mammaglobin mRNA was positive. We conclude that Mammaglobin RQ-PCR might be a useful tool for detection of leukapheresis contamination. Bone Marrow Transplantation (2001) 27, 517–523.

Objective During megakaryocyte differentiation, the immature megakaryocyte increases its ploidy to a 2 x DNA content by a process called endomitosis. This leads to the formation of a giant cell, the mature megakaryocyte, which gives rise to platelets. We investigated the role of human-nuc (h-nuc), a gene involved in septum formation in karyokynesis in yeast, during megakaryocytic polyploidization. Materials and Methods Nocodazole and 12-O-tetradecanoylphorbol-13-acetate (TPA) were used to induce megakaryocytic differentiation in K562 cell line. The ploidy distribution and CD41 expression of treated K562 cells were evaluated by flow cytometry. Using quantitative reverse transcriptase polymerase chain reaction (RT-PCR), we analyzed the h-nuc mRNA expression on treated K562 cells. Results Mature megakaryocyte-like polyploid cells were detected at day 5–7 of treatment with nocodazole. TPA also had a similar effect on K562 cells, but it was much weaker than that of nocodazole. The analysis of ploidy of nocodazole-treated K562 cells showed that nocodazole preferentially induced polyploidization of K562 cell line with a pronounced increase of the cells 8N at day 7 of culture. Expression of CD41, a differentiation-related phenotype, was significantly induced by TPA after 7 days of treatment, showing that functional maturation was mainly induced by TPA. In contrast, there was no significant increase in CD41 expression in nocodazole-treated K562 cells, suggesting that polyploidization and functional maturation are separately regulated during megakaryocytopoiesis. RT-PCR analysis indicated that h-nuc mRNA increased after 72 hours in the presence of nocodazole, preceding the induction of polyploidization. Conclusions Our data indicate that h-nuc might play a role in polyploidization during megakaryocytic differentiation via inhibition of septum formation.